| According to the World Health Organization,422 million people（about5%of the world’s population）suffer from diabetes.Diabetes is the seventh leading cause of death in the world.Diabetes is usually attributed to abnormal pancreas function,which is unable to produce sufficient amounts of insulin（type I）or is caused by abnormal cells that do not respond or even resist insulin（type II）.Both pathophysiological diseases may lead to chronic hyperglycemia,leading to various metabolic abnormalities.If hyperglycemia persists,it may cause tissue damage and organ failure,inducing various complications including cardiovascular disease,blindness,risk of amputation,kidney failure,etc.,and thereby increasing mortality.Therefore,continuous monitoring of plasma levels and timely medical intervention are very important for diabetic patients.The concentration of glucose in the blood is a key parameter of the health indicators of diabetic patients:maintaining it at an appropriate level can repeat the occurrence and development of these complications.Therefore,it is very important to accurately measure blood glucose levels.Since the 1940s,lactate has played an increasingly important role in clinical diagnosis and can be considered as the second low molecular weight metabolite after glucose.Lactate is a product of glycolysis and anaerobic glucose metabolism.It is considered as a marker of hypoxia.It is also a potential indicator of stress ischemia,panic disorder and cystic fibrosis.Traditionally,blood lactate levels have been used as a training indicator to monitor athletes’best performance.The normal（basic）blood lactate concentration ranges from 0.5 to 2.2 mmol L-1,but the concentration can rise to more than 15 mmol L-1 during strenuous exercise.At present,most clinical medical diagnosis relies on blood analysis,which in turn relies on complex and time-consuming large instruments in centralized laboratories.The blood test is an invasive method for collecting samples,after which centrifugation is used to separate the plasma and chemical analysis is subsequently performed.The traditional centralized medical model requires patients to go to the hospital for treatment,which may cause adverse effects due to delayed diagnosis and treatment,especially in emergency situations.Most of health care monitorings are carried out through invasive diagnostic tools,which hinder the real-time tracking of an individual’s precise physiological health status.In order to be able to continuously monitor the health of patients for follow-up prognosis and treatment,it is essential to rely on wearable,skin-connected health tracking monitoring equipment.Recently,the emergence of the Internet of Things has promoted the development of smart wearable devices.Wearable biosensors can perform non-invasive monitoring of physiologically relevant biomarkers in biological fluids and track health status in real time.This body fluid biomarker analysis facilitates accurate sampling,pre-concentration,and avoids complicated pre-treatment procedures.These wearable monitoring devices avoid the pain caused by skin puncture,after all,invasive blood sampling is painful for infants and the elderly.Moreover,this type of equipment can provide timely alerts for the wearer’s health by continuously monitoring important information related to physiology,which is very useful for seeking timely preventive measures and clinical guidance.The combination of multiple sweat analysis and wireless signal transmission can provide important information about the wearer’s health and physical function.Based on this research background,this paper is focused on constructing a flexible wearable electrochemical biosensor and appling it to real-time monitor glucose and lactate in sweat.The work is mainly divided into the following two parts:Part 1:Construction of a flexible wearable electrochemical biosensorIn order to make the sensor fit well to the skin surface,we chose polyethylene naphthalate（PEN）as a flexible substrate to construct a flexible wearable electrochemical sensing device.The preparation process of the flexible electrode could be roughly divided into the following stages:gel casting-glue baking-photolithography-development-fixing-coating-peeling.Photolithography was first used to pattern Au electrodes and pads on the polyethylene naphthalate（PEN）subtrate.Then,layers of Ti（5 nm）/Au（60 nm）were deposited using an electron beam evaporator.Photolithography was then used to pattern Ag electrodes,followed by A 200 nm layer of Ag deposition using an electron beam evaporator.In addition,to fabricate the Ag/Ag Cl electrode,the electrode coated with Ag was immersed in 3 M KCl solution through a chlorination process by chronopotentiometry.In order to connect the as-prepared biosensor to the electrochemical workstation,silver conductive paint was used to bring the bottom end of each electrode into contact with the copper wire.Finally,an insulated nail polish（epoxy resin）was coated on the joint part.Two working electrodes were then used to detect glucose and lactic acid respectively.In order to realize the wearable and sensitive detection,we deposited gold nanopine needles（Au NNs）on the Au electrodes in order to amplify the detection signal and improve the sensor’s sensitivity.The gold nano-pine needles were prepared by electrochemical deposition by immersing the electrode in a solution of 3 mg ml-1HAu Cl4（containing Na Cl）.In order to verify the successful preparation of gold nanostructures,we carried out a series of characterization experiments.Through field emission scanning electron microscopy,we could see the morphologies of gold nanostructures prepared under different electrodeposition conditions,which were gold nano-blocks（-0.2V,900s）,gold nanorods（-0.3V,900s）and gold nano-pine needles（-0.3V,1200s）,respectively.Under a transmission electron microscope,it could be seen that the gold nano-pine needles were composed of gold nanoparticles with a diameter of about 20 nm.Through X-ray diffraction analysis,the crystal plane spacing and crystal size of the gold nanostructures could be seen.By X-ray photoelectron spectroscopy,the valence state of the gold nanostructures deposited on the flexible electrode could be derived.In the preparation process of flexible wearable electrochemical sensors,the immobilization of enzymes is a crucial step.In order to enable glucose oxidase and lactate oxidase to be well fixed on the surface of the flexible electrode and maintain high enzyme activity,we used polyethylene glycol diglycidyl ether（PEGDE）as a cross-linking agent to combine the oxidase with the electrodes.PEGDE contains two epoxy groups,which can react with the amino groups present in the enzyme protein to form a matrix on which the enzyme is immobilized.In order to verify that glucose oxidase and lactate oxidase were successfully immobilized on the surface of the flexible electrode,we carried out a series of electrochemical tests.By cyclic voltammetry analysis of glucose standard solutions and lactic acid standard solutions of different concentrations,we could see that with the increase of glucose concentration and lactic acid concentration,the peak current also increased,showing that the electron transfer increased and the current increased due to the catalytic oxidation of substrate by glucose oxidase and lactate oxidase.This indicates that glucose oxidase and lactate oxidase were successfully immobilized on the electrode surface.In order to check the reproducibility of the sensor,we selected 5 different glucose sensors to measure 50,100,and 150μM glucose solutions,and at the same time,we selected 5 different lactic acid sensors to measure 5,10,and 15m M lactic acid solutions.The results show that the sensor has good repeatability.In addition,in order to verify the stability of the biosensor,we tested the electrochemical response of the sensor to 100μM glucose solution and 5 m M lactic acid solution within 4 weeks.The electrochemical signal did not significantly decrease,proving that the biosensor was very stable.The selectivity of the Au NNs-based sweat biosensor was further investigated by I-T curve..Some electrochemically active molecules present in human sweat（such as UA,AA,Na Cl and urea）were selected to test if they interfere the response signal toward glucose and lactate.Compared with the response signals of the target molecules（glucose and lactic acid）,the detected interference signals were negligible,indicating that the sensor we prepared has good selectivity.We also carried out the flexibility test of the sensor and studied the performance of each sensor after bending and deformation to prove its good flexibility.The flexibility of the electrode was tested by 1000 continuous bending/releasing cycles in the 100μM glucose solution.And further quantitative analysis of bending variation was investigated.We bent the flexible electrode to an angle that fit the human body and performed real-time detection of the electrode in this state.The results show that the sensor has good flexibility,indicating that the sensor has the potential to be wearable,and has broad application prospects in actual wearable biological diagnosis in the future.The constructed flexible wearable electrochemical biosensor can be applied to the next step of sweat detection.Part 2:Flexible wearable electrochemical biosensor detects glucose and lactate in sweatWe applied the previously constructed flexible wearable electrochemical biosensor to the detection of glucose and lactic acid in sweat.We first collected sweat samples from 5 healthy volunteers（collected through exercise sweat）and stored them in a refrigerator at 4°C.Then the chronoamperometry was used to detect glucose and lactic acid in sweat with the prepared sensor.In order to verify the accuracy of the sensor we prepared,we used a commercial glucose and lactic acid detection kit for analysis,and detected it with an ultraviolet spectrophotometer.It was found that the detection result of the sensor was very similar to the detection result of the kit.The flexible wearable electrochemical biosensor prepared by us can perform well for sweat analysis. |
PDF Full Text Request